Promoters viz. Cauliflower Mosaic Virus 35S (CaMV35S) and its variant, CaMV35S double enhancer, Figwort Mosaic Virus (FMV) and its variant, FMV double enhancer and others have been used for high level constitutive expression of heterologous genes in transgenic plant systems. The choice of a promoter is largely based on the required expression level(s) of the gene(s) under consideration. In case of marker genes, a threshold level of expression is necessary to enable selection of transgenic plants in vitro and/or in vivo. Among all homologous and heterologous promoters studied for constitutive expression of transgenes in plants, the CaMV35S and its variant, CaMV35S double enhancer promoters are known to induce significantly high levels of expression and are therefore widely used in transgenic plant research. Both these promoters are also characterized by strong enhancing functions and have been shown to induce a 40 to 50-fold increase in transcription of neighboring genes (Kay et al 1987, Science 236:1299-1302). The use of CaMV35S promoter for expression of a particular gene would therefore also influence expression of other genes present within the same transformation vector. This is of particular concern when one requires regulated expression of a gene by its transcriptional control under a tissue-specific promoter along with constitutive expression of a marker gene under transcriptional control of a strong constitutive promoter, both being located in the same DNA construct used for development of transgenic plants. Furthermore, presence of a strong promoter with enhancing effects could also lead to deceptive results in studies on temporal and spatial expression patterns of tissue-specific promoters in transgenic plant systems.
Production of male-sterile lines is important for development of hybrids in crop plants to enhance crop productivity. Use of hybrids for increasing crop yield is primarily based on utilization of the phenomenon of hybrid vigor or heterosis (Shull 1952, In Heterosis, Gowen Ed. 14-48 Ames: Iwo State College Press). Hybrid vigor has been exploited in plant breeding for several years. When two genetically diverse parents with compensatory agronomic characters are combined by conventional breeding methodologies, the F1 hybrid plants show higher yield than either of the parents [William 1959, Nature (Lond.) 184:527-530; Sinha and Khanna 1975, Adv. Agron. 27:123-174; Pradhan et al. 1993, Euphytica 69:219-229]. To produce hybrid progeny, cross-pollination must occur. However, there are several crop plants, which are naturally self-pollinating, for instance Brassica sp., rice and wheat. In the event of parental lines being self-pollinating, either the pollen-producing organ (anthers) must be removed or the male reproductive units (microspores) must be destroyed in one parent to facilitate cross-pollination. It is in this context, that the development of stable, normal male sterile lines assumes importance.
A simple method of generating a male sterile line is by physical removal of anthers (emasculation). Hybrids in maize, cotton and tomato are produced by this method.
Another approach for generation of male-sterile lines for hybrid seed production is the use of cytoplasmic male sterility (CMS) systems. CMS is a maternally inherited phenomenon manifesting itself as the inability to produce functional pollen grains. The genetic determinants of male sterility in CMS systems are located in genomes of the cytoplasmic organelles, the mitochondria. Restorer genes for CMS systems are dominant nuclear genes that suppress male-sterile effects of the cytoplasm (mitochondria). When incorporated into the male parent, they can function as restorers of male fertility in the F1 hybrids. CMS systems have found widespread use in the production of hybrids in sorghum, sunflower, pearl millet and sugarbeet. However, their use has been limited in corn, wheat and oilseed Brassicas due to linkage of undesirable traits such as increased disease susceptibility, chlorosis, distortion of petals, poor nectary function, etc. with CMS in these systems (McVetty et al. 1989, Can. J. Plant Sci. 69:915-918; Burns et al 1991, Can. J Plant Sci. 71:655-661; Williams 1995, Trends Biotech. 13:344-349; Buzza 1995, In Brassica oilseeds: Production and Utilisation, Kimber and McGregor, Eds. CAB International).
With the advent of recombinant DNA and plant transformation technologies, pollination control based on genetic engineering of nuclear male sterility has emerged as a tangible option for production of male-sterile plants (reviewed by Williams 1995, Trends Biotech. 13:344-349). In these methods, the plant is provided with a male-sterility gene comprising a DNA sequence coding, for example, a cytotoxic product. The cytotoxic product, in many cases, may be a lethal gene under transcriptional control of a promoter, which is predominantly active in selective tissue(s) of the male reproductive organs in plants. As an example, male sterility could be successfully induced in transgenic tobacco and oilseed rape (Brassica napus) by targeted expression of a ribonuclease [barnase from Bacillus amyloliquefaciens (Hartley 1989, Trends Biochem. Sciences 14:450-454) or Rnase T1 from Aspergillus oryzae] in the tapetal tissues of anthers using a tapetum-specific promoter, TA29, from tobacco (Mariani et al 1990, Nature 347:737-741). Tapetum, which forms the innermost layers of the anther wall, is one of the most important tissues associated with pollen development. Disruption of tapetal cells by the expression of toxic proteins consequently impairs pollen development leading to male sterile plants. Several other strategies for disruption of normal pollen development have subsequently been developed (reviewed by Williams 1995, Trends Biotech. 13:344-349).
The DNA constructs used for development of male sterile lines also require the presence of a selectable marker gene(s) for in vitro selection of transformed tissues and field selection of segregants that contain the male sterility-inducing gene. Use of a strong constitutive promoter to express the marker gene is therefore important for enabling selection of transgenic plants. However, use of a strong constitutive promoter with a lethal gene in the vicinity can be detrimental to the process of generating transgenic plants using the latter because enhancing functions of the strong constitutive promoter could induce deregulated expression of the (lethal) gene that is otherwise under transcriptional control of a known tissue-specific promoter. Some DNA sequences [Scaffold or Matrix Attachment Regions (SARs/MARs)] are known to buffer, to some extent, influences of surrounding regions on transgene expression in plants (Breyne et al 1992, Plant Cell 4:463-471, Mlynarova et al 1994, Plant Cell 6:417-426, Mlynarova et al 1996, Plant Cell 8:1589-1599). However, SARs are also known to possess enhancing functions (Steif et al 1989, Nature 341:343-345, Allen et al 1993, Plant Cell 5:603-613). Use of such sequences to achieve conditional expression of lethal genes is therefore not advisable.
Plant Genetic Systems have described, in EP 0344029A1, use of the barnase gene for generation of male-sterile lines for hybrid seed production. According to this patent, the barnase construct used for plant transformation contained three components:
1. a male sterility-conferring transcription unit (TA29 promoter-barnase gene),
2. a nos promoter-nptII marker gene cassette: the nptII gene encodes the enzyme neomycin phosphotransferase which confers resistance to the antibiotic, kanamycin and can therefore be used for in vitro selection of transformed tissues, and
3. a rbcs promoter-bar marker gene cassette: the bar gene (from Streptomyces hygroscopicus) encodes the enzyme phosphinothricin acetyl transferase which confers resistance to the herbicide, Basta. It can therefore be used for field selection of lines with the male sterility-conferring gene among segregants. The rbcs promoter used in the above construct was isolated from Arabidopsis and regulates expression of the rubisco small subunit gene in the same.
Although the above patent claims capability of in vitro selection of transformed tissues on the herbicide, it is known that the rbcs promoter is highly active only in green tissues of intact plants. Its activity in iii vitro grown callus tissues is very low. Hence, it might be inadequate for in vitro selection of transgenic plants on the selective agent i.e. herbicide. The strategy described in the above patent envisages the use of two different marker genes, one primarily for in vitro selection (nptII) and another for field selection (bar). Keeping in mind the general reluctance to accept marker genes conferring resistance to antibiotics, the presence of nos-nptII in the transgenic lines is superfluous. Further, there is no information in the said patent on transformation frequencies of plants obtained using the barnase gene or other constructs containing any other lethal gene. Therefore, the ease of developing transgenic plants with conditional expression of lethal genes has not been described. Moreover, the barnase gene construct described in the above patent does not envisage the need for the inhibitor gene, barstar in the background (as evidenced in this study and by Paul et al 1992, Plant Mol. Biol. 19:611-622). Barstar is an inhibitor protein produced by the bacterium, Bacillus amyloliquefaciens, wherein it negates the lethal effects of the ribonuclease by forming a one-to-one complex with the same in the cytoplasm (Hartley and Smeaton 0.1973, J. Biochem. 248:5624-5626).
Forbio Research Pvt. Ltd. in WO9730162A1 has described a method for protecting tissues from leaky expression of barnase gene by using coordinated expression of two additional components. One of these components is the inhibitor gene, barstar under transcriptional control of a modified CaMV35S promoter containing the repressor-binding site (operator) of the lac operon of the bacterium, Escherichia coli. Expression of the barstar gene, in turn is regulated by a second modulator gene encoding the repressor of the lac operon (lacIq) expressed under the same tissue-specific promoter as the barnase gene. The above strategy is a multi-component, multi-functional system, which necessitates proper functionality of each component. Any variation in expression of the same can render the entire system non-functional. Moreover, the strategy also envisages the use of many redundant genes and promoters, the presence of which is undesirable in agronomic applications of the said technology.
Plant Genetic System, in a subsequent U.S. Pat. No. 6,025,546, has described another strategy for development of male sterile plants in corn, rice and oilseed rape (Brassica napus) using the barnase gene under transcriptional control of appropriate tapetum-specific promoter(s). This patent also describes use of the inhibitor gene, barstar as a co-regulator for development of male sterile lines. Further, it highlights the need to clone the barstar gene in the background of all barnase-containing vectors. The barstar gene is preferentially placed under transcriptional control of a constitutive promoter (for example, CaMV35S promoter) to negate any undesirable effects on transformed tissues due to leaky expression of the barnase gene. The co-regulating gene (barstar) may be located in the same transformation vector containing the barnase gene or may be used independently in co-transformation experiments. In addition, the applicants of this patent describe yet another strategy involving deployment of the barstar gene under control of a minimal promoter in order to utilize enhancing functions of plant enhancers. According to the patent, these strategies would not only be effective in countering leaky expression of the barnase gene due to position effects but would also increase the frequency of male sterile plants exhibiting good agronomic performance. Although the patent highlights the fact that the described strategy could be used for any sterility-inducing DNA, it follows that such a strategy would be effective only in situations wherein a corresponding inhibitor protein is known for the male-sterility gene.
Problems associated with leaky or deregulated expression of tissue-specific promoters in the presence of a strong constitutive promoter are, therefore, a major impediment to targeted expression of genes in specific tissues. The present invention addresses the above issue without the involvement of any co-regulating gene or other functional component(s). Further, it facilitates development of strategies for producing stable and normal male sterile lines at high frequencies to exploit the development in genetic engineering technologies and genomic research for improving yields of crop plants. The present invention provides a strategy for protecting tissue-specific expression using one of the strongest constitutive promoters and one of the most potent lethality-inducing genes known to date merely as an example. The strategy involves use of a DNA sequence, hereinafter referred to as xe2x80x9cInsulatorxe2x80x9d, which distances the strong constitutive promoter from the tissue specific promoter in a construct used for development of transgenic plants. The efficacy of the strategy would be applicable to any other sequence combination with similar properties in transgenic plants.
The main object of the invention is to provide a novel Insulator construct for protecting tissue-specific expression of a lethal gene from enhancing functions of a strong constitutive promoter located in the vicinity thereof.
Another object is to develop a construct for controlling leaky expression of a lethal gene, said construct containing a single selectable marker gene for in vitro as well as field-level selection of transgenic plants.
A further object is to develop a construct that can be used for controlling leaky expression of a lethal gene without the involvement of any other regulatory or inhibitor sequence component.
Another object is to develop a construct that would confer protection against leaky expression of a lethal gene over all the developmental stages of a plant.
Yet another object is to develop normal, stable male sterile lines generated at a high frequency by genetic transformation using constructs developed according to the invention to protect tissue-specific expression of a lethal gene.
Still another object is to provide methods for developing normal, stable male sterile lines generated at a high frequency in crop plants using the Insulator construct of the invention.
The invention provides a novel Insulator construct for controlling leaky expression of a lethal gene from enhancing functions of a strong constitutive promoter present in the said Insulator construct following integration into the genome of a plant, said construct comprising (i) a first transcription unit containing a lethal gene under transcriptional control of a tapetum-specific promoter and fused to a suitable transcription termination signal including a polyadenylation signal, (ii) a second transcriptional unit comprising a selectable marker gene, under transcriptional control of a strong constitutive promoter with a leader sequence and fused to a suitable transcription termination signal, including a polyadenylation signal and (iii) an Insulator sequence placed between the first and second transcription units, so as to distance the first transcription unit from enhancing functions of the constitutively expressing promoter in the second transcription unit. Further, the invention provides a method for development of stable, normal male sterile lines using the Insulator construct, the said method comprising a series of checks or xe2x80x9csievesxe2x80x9d to test for protected tissue-specific expression of the lethal gene in the presence of the strong constitutive promoter. The said method comprises the steps of testing transformation and regeneration frequencies, development of male sterile transgenic plants, analysis of vegetative morphology and female fertility of male sterile plants, Southern analysis for identification of male sterile plants containing a single copy of the T-DNA insert, analysis of germination frequencies and segregation data of T1 seeds and stable inheritance of male sterility in T1 progeny.
The present invention relates to an Insulator construct for controlling leaky expression of a lethal gene from enhancing functions of a strong constitutive promoter present in the said Insulator construct following integration into the genome of a plant and a method for development of male sterile lines in crop plants using the said Insulator construct.
Accordingly, the invention provides an Insulator construct comprising:
i) a first transcription unit comprising a lethal gene under transcriptional control of a tissue specific promoter for targeted expression in specific tissue(s) and fused to a suitable transcription termination signal, including a polyadenylation signal,
ii) a second transcription unit comprising a selectable marker gene under transcriptional control of a strong constitutive promoter with a leader sequence and fused to a suitable transcription termination signal, including a polyadenylation signal, and
iii) an Insulator sequence placed between the first and second transcription units so as to distance the first transcription unit from enhancing functions of the constitutively expressing promoter in the second transcription unit.
In an embodiment, the lethal genes that can be used in the Insulator construct could be any coding sequence which, when expressed in a cell (as RNA or protein), significantly disrupts the normal metabolism, functioning or development of any such cell, preferably leading thereby to death of the cell. Instances of such lethal genes include ribonucleases such as RNAseT1 (from Aspergillus oryzae), barnase (from Bacillus amyloliquefaciens), binase (from Bacillus intermedius); rol genes from Agrobacterium rhizogenes; diphtheria toxin A chain-encoding gene etc. In a preferred embodiment, the lethal gene used in the Insulator construct is the ribonuclease gene, barnase derived from the bacterium Bacillus amyloliquefaciens. 
Tissue specific promoters are characterized by definite temporal and spatial expression patterns during plant growth and development. A large number of such promoters are known in plant systems. A few examples include anther specific promoters such as TA29, A9, tap1, bcp1 or seed specific promoters such as napin. In an embodiment the tissue specific promoter used to express the barnase gene is the tapetum-specific promoter TA29.
A selectable marker gene encodes a RNA or protein which, when expressed in the cells of the plant, gives the cells expressing the gene a selective advantage over cells lacking the same. In an embodiment, the marker gene is selected from the group of herbicide resistance-conferring genes such as bar, tfdA, ALS; antibiotic resistance-conferring genes such as nptII, hpt, aadA, etc. In a preferred embodiment of the invention, the selectable marker gene used for in vitro and in vivo selection of transformed tissues is the herbicide resistance conferring gene, bar from Streptomyces hygroscopicus. 
In an embodiment, the promoter used for driving the expression of the bar gene is the CaMV35S promoter, which is a known strong constitutive promoter.
A leader sequence of the Alfalfa Mosaic Virus (AMV) RNA4 gene has been incorporated in the above constructs between the CaMV35S promoter and the bar gene. Such leader sequences are known to enhance expression levels by improving translational efficiency without influencing promoter strength (Gallie et al 1987, Nucl. Acids Res. 15:8693-8711, Day et al 1993, Plant Mol. Biol. 23:97-109).
The transcription unit of the selectable marker gene is placed towards the Left Border of T-DNA (in an appropriate binary vector) to ensure complete transfer of all components located between the T-DNA borders during Agrobacterium-mediated genetic transformation.
The Insulator sequence comprises a sequence derived from genomic DNA of a plant. It does not encode any functional RNA or protein but when placed between the two transcription units of the Insulator construct, serves to substantially negate the deleterious effects resulting from leaky expression of the lethal gene due to enhancing functions of the strong constitutive promoter. While there are no limitations as to the sequence per se that can be used as an Insulator, it is recommended that a sequence with the following properties be selected:
i) The Insulator sequence should not bear strict homology with any component of the host genome in order to avoid, to the maximum extent possible, induction of homology-dependent gene silencing (reviewed in Meyer and Saedler 1996, Ann. Rev. Plant Physiol. Plant Mol. Biol. 47:23-48).
ii) GC content of the Insulator sequence should be in consonance with transcriptionally active regions of the host genome.
iii) The Insulator sequence should not encode any functional or regulatory component nor possess any regulatory or enhancer elements or sequences that may influence expression of neighboring genes.
In still another embodiment, the Insulator sequence comprises a DNA sequence generated from partial coding sequences of two dicot genes (a topoisomerase gene from pea and an acetolactate synthase gene from Arabidopsis).
The length of the Insulator sequence used in the Insulator construct would vary according to the nature and strength of the constitutive promoter used. The optimum length of the Insulator sequence is governed by the primary objective of generating a large number of normal male sterile plants. It is recommended that the Insulator sequence may have a length of at least 2 kb. In a preferred embodiment, the Insulator sequence has a length of about 5 kb.
DNA vector(s) containing the barnase gene also have the barstar gene transcribed by its native bacterial promoter cloned in the vector backbone. This was necessitated because of the inability to clone the barnase gene in the absence of background levels of the Barstar protein. The binary vector, pPZP200 (Hajdukiewicz et al 1994, Plant Mol. Biol. 25:989-994), was used as the transformation vector.
The invention also provides a method to obtain normal male-sterile plants at a high frequency. The said method incorporates a series of checks or xe2x80x9csievesxe2x80x9d to test for protected tissue-specific expression of the lethal gene in the presence of the strong constitutive promoter. The said sieves are primarily identified and selected for on the basis of their role in determining the agronomic viability of any (transgenic) crop plant and are as outlined below:
i) frequency of genetic transformation and regeneration
ii) vegetative morphology of transgenic male sterile plants
iii) female fertility of transgenic male sterile plants
iv) germination frequencies of T1 seeds obtained by backcrossing transgenic male sterile plants
v) segregation ratios of marker gene/male sterility among T1 plants
vi) stable inheritance of male sterile phenotype among T1 plants.
The said method for the development of male sterile plants comprises the steps of:
i) transforming the nuclear genome of plant cells with an Insulator construct comprising:
a) a first transcription unit comprising a lethal gene under transcriptional control of a tissue specific promoter for targeted expression in specific tissue(s) and fused to a suitable transcription termination signal, including a polyadenylation signal,
b) a second transcription unit comprising a selectable marker gene under transcriptional control of a strong constitutive promoter with a leader sequence and fused to a suitable transcription termination signal, including a polyadenylation signal,
c) an Insulator sequence placed between the first and second transcription units so as to distance the first transcription unit from enhancing influences of the constitutive promoter in the second transcription unit.
ii) regenerating plants from said transformed plant cells,
iii) identification of male sterile transgenic plants by morphological observations and by their failure to set seed on selfing,
iv) obtaining, at a high frequency, male sterile plants with normal vegetative morphology and normal female fertility,
v) identifying single copy male sterile lines by Southern hybridization,
vi) back-crossing male sterile plants with untransformed parent to obtain T1 seeds,
vii) obtaining male sterile plants with normal T1 seed germination frequencies,
viii) obtaining normal segregation ratio of marker gene among T1 progeny of single copy male sterile plants identified,
ix) obtaining stable transfer of male sterile phenotype among all T1 plants exhibiting marker resistance.
In an embodiment, the preferred lethal gene is barnase.
In still another embodiment, the preferred tissue specific promoter is TA29.
In yet another embodiment, the preferred marker gene is bar.
In another embodiment, the preferred constitutive promoter is CaMV35S promoter.
In an embodiment, the Insulator sequence has a length of about 5 kb.
In an embodiment, the crop plants used for genetic transformation are selected from dicotyledonous plants, such as Brassica juncea. 
In another embodiment, the procedure used for development of transformed plants in Brassica juncea is Agrobacterium-mediated transformation using disarmed Ti plasmid.
In an embodiment, the transgenic male-sterile plants are scored for abnormalities in vegetative characters
In another embodiment, the male sterile transgenic plants are backcrossed to the untransformed parent and tested for female fertility.
In another embodiment, the male sterile plants are analyzed by Southern hybridization to identify transgenic plants containing a single copy of the T-DNA insert.
In yet another embodiment, seeds obtained from backcrossing the above male sterile plants are tested for their viability as evidenced by their ability to germinate on non-selective media.
In still another embodiment, germinated seedlings obtained from backcrossed seeds were tested for segregation of the marker gene by transferring them on selective media.
In another embodiment, the T1 plants obtained from selected backcrossed progeny were transferred to field conditions and tested for stable inheritance of the male sterile phenotype.
The invention is described in detail hereinafter, with reference to the accompanying drawings and examples. Various modifications, especially with respect to the Insulator construct and the methods used to deploy the same would be obvious to those skilled in the art. Such modifications are deemed to fall within the scope of the present invention and the examples and embodiments provided herein should not be construed as limitations on the inventive concept embodied in this invention.